Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression

Abstract

Glucocorticoids are the most effective antiinflammatory agents for the treatment of chronic inflammatory diseases even though some diseases, such as chronic obstructive pulmonary disease (COPD), are relatively glucocorticoid insensitive. However, the molecular mechanism of this glucocorticoid insensitivity remains uncertain. We show that a defect of glucocorticoid receptor (GR) deacetylation caused by impaired histone deacetylase (HDAC) 2 induces glucocorticoid insensitivity toward nuclear factor (NF)-kappaB-mediated gene expression. Specific knockdown of HDAC2 by RNA interference resulted in reduced sensitivity to dexamethasone suppression of interleukin 1beta-induced granulocyte/macrophage colony-stimulating factor production. Loss of HDAC2 did not reduce GR nuclear translocation, GR binding to glucocorticoid response element (GRE) on DNA, or GR-induced DNA or gene induction but inhibited the association between GR and NF-kappaB. GR becomes acetylated after ligand binding, and HDAC2-mediated GR deacetylation enables GR binding to the NF-kappaB complex. Site-directed mutagenesis of K494 and K495 reduced GR acetylation, and the ability to repress NF-kappaB-dependent gene expression becomes insensitive to histone deacetylase inhibition. In conclusion, we show that overexpression of HDAC2 in glucocorticoid-insensitive alveolar macrophages from patients with COPD is able to restore glucocorticoid sensitivity. Thus, reduction of HDAC2 plays a critical role in glucocorticoid insensitivity in repressing NF-kappaB-mediated, but not GRE-mediated, gene expression.

Figure 1.

HDAC2 KD-induced glucocorticoid insensitivity in A549 cells. (A) Dot blot analysis of nuclear extracts from A549 cells 48 h after transfection with siRNA against HDAC1, -2, -3, and -8; Sc; or vehicle only (NT). (B) IL-1β–stimulated (1 ng/ml for 24 h) GM-CSF production. Values represent means ± SEM. * and **, P < 0.05 and P < 0.01, respectively, versus IL-1β control; #, P < 0.05 versus basal control. n = 4∼8 experiments. (C) Concentration response curve of Dex suppression of IL-1β–induced GM-CSF production, and (D) summarized EC₅₀ values of Dex (n = 4∼8). Values in C represent means ± SEM. (E) Correlation between EC₅₀ and HDAC2 expression in HDAC2 RNAi cells. Different levels of HDAC2 expression were induced using individual siRNA or combinations of up to four duplexes against HDAC2. HDAC2 expression evaluated by Western blotting was shown as the percentage of that in nontreated cells. Dashed and continuous lines represent 95% confidence interval.

Figure 2.

HDAC2 KD does not inhibit GR-mediated gene activation. (A) GR nuclear translocation in Sc and HDAC2 siRNAs (H2) transfected cells stimulated with 10⁻⁸ M Dex for 1 h. β-Actin and histone H1 were used as loading controls. GR-GRE binding (B), GRE-luciferase activity (C), and SLPI gene induction (D). * and **, P < 0.05 and P < 0.01, respectively (n = 3 experiments). NT, nontreated. (E) GR-GRE binding 1 h (closed circle) and 8 h (open circle) after Dex stimulation. Values in B–E represent means ± SEM.

Figure 3.

HDAC2 KD causes inhibition of GR–NF-κB association. (A) GR and HDAC2 were coimmunoprecipitated with p65–NF-κB in nuclear extracts in the presence or absence of a 30-min treatment of IL-1β with or without 20-min pretreatment with 10⁻⁹ M Dex. H2, HDAC2. (B) NF-κB activation measured by NF-κB binding to oligonucleotides, including NF-κB binding site, 30 min after IL-1β treatment (n = 3). (C) Histone 4 acetylation of NF-κB binding site at the GM-CSF promoter region detected by ChIP assay. Ct (threshold) values of PCR products were normalized to those of input samples. *, P < 0.05 versus IL-1β control (n = 3 experiments). Values in B and C represent means ± SEM.

Figure 4.

GR deacetylation by HDAC2 is a prerequisite for p65–NF-κB binding. (A) GR was precipitated after vehicle or Dex treatment with anti-GR antibody in whole cell extracts and with GRE oligonucleotides or anti-p65–NF-κB antibody in the presence of 10⁻⁸ M Dex and 1 ng/ml IL-1β. Bands were visualized by anti–acetyl-lysine antibody (α-AcK) and anti-GR antibody (α-GR). (B) Graphical representation of the results shown in A, with the ratio of AcK band to GR band in nontreated (shaded bar) and HDAC2 RNAi cells (closed bar). X represents no GR recruitment to NF-κB. *, P < 0.05. (C) GR acetylation level of each site-directed mutant after treatment with 10⁻⁶ M Dex for 1 h. GR were pulled down with anti–His-tag antibody. (D) SLPI mRNA level after treatment with 10⁻⁶ M Dex for 4 h. (E) GR binding to p65 under treatment with 10⁻⁸ M Dex for 1 h. GR is immunoprecipitated with His-tag antibody 1 h after IL-1β treatment. (F) Effect of 10⁻⁸ M Dex on IL-1β–induced GM-CSF production in the presence of 10 nM TSA for 10 min. *, P < 0.05. (G) AcK detection on immunoprecipitated GR in the presence of 10⁻⁶ M Dex after incubation with HDAC1, -2, -3, or -8 for 4 h at 30(C. (bottom) The ratio of AcK band to GR band is shown graphically. * and **, P < 0.05 and P < 0.01, respectively, versus control (n = 3 experiments). Values in B, D, F, and G represent means ± SEM.

Figure 5.

Overexpression of HDAC2 restores glucocorticoid sensitivity in alveolar macrophages from COPD patients. (A) Total HDAC activity (closed bar) and immunoprecipitated HDAC2 (shaded bar) activity in nuclear extracts. * and **, P < 0.05 and P < 0.001, respectively versus healthy nonsmokers; #, P < 0.05 versus smokers. (B) Representative image of HDAC2 protein expression in nuclear extracts 24 h after transfection with vehicle (NT), empty vector (Em), and HDAC2 vector (H2). (C) Acetylation level of immunoprecipitated GR of alveolar macrophages from normal (N), smoker (S), and COPD patients (C). Cells were stimulated with 10⁻⁸ M Dex for 1 h. (D) 100 ng/ml LPS-induced GM-CSF production in the absence (closed bar) or presence (shaded bar) of 10⁻⁸ M Dex (for 20 min) 24 h after transfection with each vector. Open bars are unstimulated control samples. *, P < 0.05 versus LPS control (n = 6 experiments). (E) LPS-induced GM-CSF production in nontreated (NT), Sc, or HDAC2 siRNA (H2)–transfected sputum macrophages in the absence (closed bar) or presence (shaded bar) of 10⁻⁸ M Dex for 20 min. *, P < 0.05. Values in A, D, and E represent means ± SEM.